See related article, pages 478 – 486
NADH, a New Player in the Cardiac Ryanodine Receptor?
Gerhard Meissner
I
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itors (rotenone, pyridaben, and antimycin A) relieve inhibition of RyR2 activity by NADH.1 The data suggest that
NADH exerts its effect via an NADH oxidase activity rather
than a direct interaction with RyR2.
In support of an action via an RyR2-associated protein,
NADH reduced RyR2 activity in the presence of MgATP,
when cardiac SR vesicles, but not purified RyRs, were fused
with lipid bilayers (L. Xu and G. Meissner, unpublished data).
Photolabeling studies with [3H](trifluoromethyl)diazirinylpyridaben revealed the presence of a mitochondrial PSSTlike 23-kDa protein in membrane fractions enriched in
RyR2.1 PSST acts as an intermediate in electron transfer in
complex I from NADH to ubiquinone. In contrast, RyR2associated NADH oxidase was not stimulated by an exogenously added ubiquinone analogue. Cardiac mitochondrial
particles exhibited a low NADH oxidase activity that was
activated by the exogenous electron acceptor, pointing to
distinct differences in the pharmacology of RyR2-associated
and mitochondrial NADH oxidases.1
Cardiac myocytes have a microsomal NADH oxidase that
is a major source of O2·⫺ production in cardiomyocytes.9 The
enzyme is regulated by PO2 and contains a diphenylene
iodonium-inhibitable flavoprotein site. Cherednichenko et al1
confirmed the presence of a diphenylene iodoniuminhibitable NADH oxidase in their cardiac SR preparation but
did not describe the effect of the inhibitor on NADH-mediated
RyR2 activity. Reactive oxygen species such as O2·⫺ and H2O2
affect SR function by modulating RyR and SR Ca2⫹ pump
activities. However, single-channel measurements showed
that NADH inhibition of RyR2 was not affected by superoxide dismutase and thus was independent on O2·⫺ production.8
Rather, O2·⫺ had a slight stimulatory effect, consistent with
observations that O2·⫺ activates RyR2.10
In skeletal muscle, the major isoform RyR1 is also modulated by an NADH oxidase. Xia et al11 found the enzyme
requires O2 but otherwise differs from the oxidase acting on
RyR2. Mitochondrial electron transport inhibitors did not
inhibit modulation by NADH. In contrast to inhibition of
RyR2, the skeletal NADH oxidase stimulated RyR1 and
activation was inhibited by superoxide dismutase, suggesting
that the enzyme that activated RyR1 produced O2·⫺. Baker et
al12 identified an N-terminal oxidoreductase-like domain in
RyR1. RyR1 bound NAD⫹ to sites other than the ATPbinding site, but it is unclear whether the oxidoreductase-like
domain is enzymatically active. Contrary to the results of Xia
et al,11 NADH had only minor effects on RyR1 activity.
The mechanism of NADH inhibition of the cardiac RyR2
ion channel activity is unclear. One possibility is that RyR2
senses changes in NADH oxidase conformation that are
controlled by NADH/NAD⫹. Alternatively, the NADH oxidase may transfer reducing equivalents to RyR2. RyR ion
channels contain regulatory thiol groups susceptible to redox-
n this issue of Circulation Research, Cherednichenko et
al1 describe an NADH oxidase activity that regulates the
ryanodine receptor ion channel (RyR2) in cardiac muscle.
Mammalian tissues express three closely related 560-kDa
ryanodine receptors (RyRs) encoded by separate genes. RyR1
is the predominant isoform in skeletal muscle, and RyR2
predominates in heart. RyR3 is widely expressed at low
levels. RyRs control diverse cellular functions by releasing
Ca2⫹ ions from intracellular membrane-bound Ca2⫹ stores. In
cardiac muscle, release of Ca2⫹ from the sarcoplasmic reticulum (SR) into the cytoplasm leads to muscle contraction.
Released Ca2⫹ returns to SR by an ATP-dependent Ca2⫹
pump, resulting in muscle relaxation. The RyRs are regulated
by myriad pathways through small diffusible molecules such
as Ca2⫹, Mg2⫹, and ATP and by calmodulin, kinases, and
phosphatases.2,3
The RyRs are also targets for redox active molecules
(Figure).3,4 Active muscle produces reactive oxygen and
reactive nitrogen species that modulate RyR2. Changes in
oxygen tension or the ratio of reduced to oxidized glutathione
modulate RyR2 activity by reducing and oxidizing cysteine
residues (J. Sun and G. Meissner, unpublished data). RyR2 is
endogenously S-nitrosylated,5 and an association of RyR2
with neuronal nitric oxide synthase has been described,6
suggesting NO and related molecules are physiological modulators of cardiac muscle excitation-contraction coupling.
The study by Cherednichenko et al1 along with two recent
reports7,8 provide evidence for an additional redox-sensing
mechanism in cardiac muscle. An NADH oxidase is shown to
modulate RyR2 through the cytosolic NADH/NAD⫹ redox
potential in cardiac myocytes.
The present report builds on an earlier observation of a
regulation of RyR2 by NADH. Zima et al7 compared the
effects of NADH, NAD⫹, and NADPH on single RyR1 and
RyR2 ion channels isolated from rabbit skeletal muscle and
rat cardiac muscle, using the planar lipid bilayer method.
RyR2 activity decreased in the presence of NADH and
increased with NAD⫹. Inhibition by NADH was reversed by
equimolar amounts of NAD⫹. NADPH was without effect.
Regulation of RyR1 by NADH via the ATP-binding site was
observed but ruled out for RyR2. A striking finding of the
present report is that mitochondrial electron transport inhibThe opinions expressed in this editorial are not necessarily those of the
editors or of the American Heart Association.
From the Departments of Biochemistry and Biophysics, and Cell and
Molecular Physiology, University of North Carolina, Chapel Hill, NC.
Correspondence to Gerhard Meissner, Department of Biochemistry
and Biophysics, University of North Carolina, Chapel Hill, NC 275997260. E-mail [email protected]
(Circ Res. 2004;94:418-419.)
© 2004 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000122072.43826.98
418
Meissner
Inhibition of RyR2 by NADH
419
Acknowledgments
This work was supported by NIH grants AR18687 and HL73051.
References
Modulation of RyR2 activity by redox active molecules.
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based modifications,3,4 suggesting that thiols modulate RyR2
activity. Future studies will establish whether NADH and
NAD⫹ affect RyR2 function via specific modulatory thiols.
The native cardiac muscle RyR2 has a large number of free
cysteines (⬎25 per 560-kDa RyR subunit) in the presence of
5 mmol/L reduced glutathione (J. Sun and G. Meissner,
unpublished data). Thus, many thiols are likely in a reduced
state in normal functioning hearts due to the reducing
environment created by thiol-reducing compounds such as
glutathione. Cherednichenko et al1 suggest that the NADH
oxidase is part of a negative-feedback mechanism that couples SR Ca2⫹ release with mitochondrial Ca2⫹ and energy
metabolism. The ratio of cytosolic NADH/NAD⫹ ratio in
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Perhaps a more important function of the NADH oxidase is
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since NAD⫹ counteracts the action of NADH.7,8 Even during
the extreme anaerobic condition of sustained ischemia, the
cytosolic NADH/NAD⫹ ratio13 was below that found to
inhibit SR Ca2⫹ release and RyR2 activity.7,8 Future therapeutic approaches might benefit from a focus on the redox
modulation of the cardiac muscle ryanodine receptor.
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KEY WORDS: cardiac muscle Ca2⫹ release
oxidase
䡲
redox modulation
䡲
NADH
NADH, a New Player in the Cardiac Ryanodine Receptor?
Gerhard Meissner
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Circ Res. 2004;94:418-419
doi: 10.1161/01.RES.0000122072.43826.98
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